T type calcium channel inhibitors

ABSTRACT

The present invention provides novel T type calcium channel inhibitors of formula (I), the use thereof in the treatment of a disease or condition in a mammal associated with influx of extracellular calcium via T type calcium channels, 
                         
wherein
         R 1  is C 1 -C 4  alkyl, hydroxy, or C 1 -C 4  alkoxy;   Z is NH, NCH 3 , O, S, or CH 2 ;   Y is NH, O, or CH 2  with the proviso that Y and Z are not the same;   R 2  is H, halo, NH 2 , C 1 -C 4  alkyl, hydroxy, or C 1 -C 4  alkoxy;   m and n are independently selected from integers ranging from 1-5 with the proviso that m+n=an integer ranging from 2-9; and   R 3  is H, halo, NH 2 , C 1 -C 4  alkyl, hydroxy, or C 1 -C 4  alkoxy.

CROSS REFERENCE TO RELATED APPLICATION

The application claims priority under 35 U.S.C. §1.119(e) of U.S.Provisional Patent Application Ser. No. 60/603,168, filed Aug. 20, 2004,the complete disclosure of which is hereby incorporated by its referencefor all purposes.

BACKGROUND OF THE INVENTION

Influx of extracellular calcium is critical for a number of vitalcellular processes. Calcium influx is generally mediated by calciumchannels, which are grouped into several families one of which is the Ttype calcium channel family. Pharmacological modulation of the T typecalcium channel's function is tremendously important in the practice ofmedicine; for example, T type calcium channel inhibitors are inwidespread use in the treatment of neurological diseases (e.g. epilepsy,petit mal seizure, absence seizure, neuropathic pain, and etc.) andcardiovascular diseases (e.g. hypertension, unstable angina, and etc.).For example, mibefradil, a T type calcium inhibitor, was clinicallyefficacious in treating hypertension and cardiac arrhythmia. Studiesalso suggest that T-type calcium channels may play an important role inage related macular degeneration. Recently, we have showed that the α1Hand δ25 isoforms of T type calcium channels are present in cancer celllines and that novel chemical agents could be synthesized to blockcalcium entry via this channel thus inhibiting cancer cellproliferation.

Ca2+ entry is critical for cellular proliferation. Because unusuallyrapid proliferation is a hallmark of cancer, elucidation of themechanism of Ca2+ entry is of scientific importance with potentialclinical significance. However, the mechanism or mechanisms of Ca2+entry in electrically non-excitable cells, which constitute most typesof cancer, remain elusive. The majority of cancers arise from cell typesconsidered “electrically non-excitable.” This is to distinguish themeans of regulation of Ca2+ entry in these cells from those havingaction potentials, that is “electrically excitable” cells. Inelectrically excitable cells, an action potential opens voltage gatedCa-2+ channels that admit the Ca2+ required for initiating events suchas neurosecretion. In electrically non-excitable cells, the regulatorymechanism activating Ca2+ entry is unclear in part because of theuncertainty about the molecular mechanism by which Ca2+ entry occurs.

The two types of ion channels or transporters most commonly implicatedin capacitative Ca+ entry are I_(CRAC) and members of the Trp. These twochannel types do not share many properties other than relativeselectivity for Ca2+, albeit sometimes very weak, and lack of voltagedependent gating. It has been difficult, however, to tie these proteins'function to capacitative Ca+ entry. This may reflect a particularcomplexity of Ca2+ signaling in electrically non-excitable cells arisingfrom participation of numerous channels and transporters in thisprocess. It is also possible that the primary means of Ca2+ entry inelectrically non-excitable cells has yet to be fully elucidated.

We have taken an alternative approach to dissecting the Ca2+ entrypathway in electrically non-excitable cells. We first took advantage ofCa+entry blockade by Ni2+, as measured by fluorescence techniques(Merritt, J. E. and Rink, T. J. 1987. Regulation of cytosolic freecalcium in fura-2-loaded rat parotid acinar cells. J. Biol. Chem.262:17362-17369; Merritt, J. E., Jacob, R., and Hallam, T. J. 1989. Useof manganese to discriminate between calcium influx and mobilizationfrom internal stores in stimulated human neutrophils. J. Biol. Chem.264:1522-1527; Skryma, R., Mariot, P., Bourhis, X. L., Coppenolle, F.V., Shuba, Y., Abeele, F. V., Legrand, G., Humez, S., Boilly, B., andPrevarskaya, N. 2000. Store depletion and store-operated Ca²⁺ current inhuman prostate cancer LNCaP cells: involvement in apoptosis. J. Physiol.(Lond.) 527 Pt 1:71-83), to identify compounds in the publishedliterature with a similar ability. The structure/activity relationshipof these known compounds was used to guide the synthesis of novelcompounds with enhanced potency to block Ca2+ entry into andproliferation of several cancer cell lines. Administration of one ofthese compounds, TH-1177, significantly extended the lifespan of nudemice inoculated with human PC3 prostate cancer cells (Haverstick, D. M.,Heady, T. N., Macdonald, T. L., and Gray, L. S. 2000. Inhibition ofhuman prostate cancer proliferation in vitro and in a mouse model by acompound synthesized to block Ca²⁺ entry. Cancer Res 60:1002-1008). Weshow here that two representative compounds block the Ca2+ currentthrough the heterologously expressed α1H isoform of T type Ca2+ channelsand inhibit proliferation of HEK293 cells stably transfected with thisisoform. These two compounds blocked the Ca2+ current and capacitativeCa2+ entry with similar potencies and identical stereoselectivity.Importantly, cell lines sensitive to our novel compounds express messagefor α1H, its δ25 splice variant, or both while a cell line resistant toour compounds does not detectably express either message.

The library of compounds we have developed has apparently broad activityagainst cancer cell lines from various tissues. They act cytostaticallyand are equally potent at inhibiting hormone sensitive and insensitivebreast and prostate cancer lines. We demonstrate here the likely targetof their anti-proliferative activity. Taken together, these observationsraise the possibility of directed chemical synthesis of compounds thatinhibit Ca2+ entry into and thereby proliferation of cancer cells.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a method for the treatmentof a disease or condition in a mammal associated with influx ofextracellular calcium via T type calcium channels, which comprisesadministering to the mammal a therapeutically effective amount of a Ttype calcium channel inhibitor, a prodrug thereof, or a pharmaceuticallyacceptable salt of said inhibitor or prodrug. Preferably, the T typecalcium channel inhibitor has a structure represented by Formula (I):

-   -   wherein    -   R₁ is C₁-C₄ alkyl, hydroxy, or C₁-C₄ alkoxy;    -   Z is NH, NCH₃, O, S, or CH₂;    -   Y is NH, O, or CH₂ with the proviso that Y and Z are not the        same;    -   R₂ is H, halo, NH₂, C₁-C₄ alkyl, hydroxy, or C₁-C₄ alkoxy;        m and n are independently selected from integers ranging from        1-5 with the proviso that m+n=an integer ranging from 2-9; and    -   R₃ is H, halo, NH₂, C₁-C₄ alkyl, hydroxy, or C₁-C₄ alkoxy.        More preferably, R₁ is hydroxy or C₁-C₄ alkoxy; Z is N or O; R₂        is H, halo, NH₂ or hydroxy; and R₃ is H. The disease or        condition is preferably selected from the group consisting of        unstable angina, hypertension, epilepsy, neuropathic pain, petit        mal seizure, absence seizure, age related macular degeneration,        cancer, and pre-cancerous condition.

The present invention also provides a method for reducing proliferationof electrically non-excitable cells, which comprises administering a Ttype calcium channel inhibitor of formula (I) as described above.

In another aspect, the present invention provides a compound of Formula(I)

wherein

-   -   R₁ is C₁-C₄ alkyl, hydroxy, or C₁-C₄ alkoxy;    -   Z is NH, NCH₃, O, S, or CH₂;    -   Y is NH, O, or CH₂ with the proviso that Y and Z are not the        same;    -   R₂ is H, halo, NH₂, C₁-C₄ alkyl, hydroxy, or C₁-C₄ alkoxy;    -   m and n are independently selected from integers ranging from        1-5 with the proviso that m+n=an integer ranging from 2-9; and    -   R₃ is H, halo, NH₂, C₁-C₄ alkyl, hydroxy, or C₁-C₄ alkoxy;    -   or a pharmaceutically acceptable salt thereof.

Preferably, R₁ is hydroxy or C₁-C₄ alkoxy; Z is N or O; R₂ is H, halo,NH₂ or hydroxy; and R₃ is H. More preferably, the compound of formula(I) is selected from the group consisting of the following compounds:

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof formula (I) as described above, a prodrug of said compound or apharmaceutically acceptable salt of said compound or prodrug; and apharmaceutically acceptable carrier, vehicle or diluent.

In another aspect, the present invention provides a method for thetreatment of cancer or pre-cancerous condition in a mammal, whichcomprises administering to the mammal a therapeutically effective amountof a compound of formula (I) as described above, a prodrug thereof, or apharmaceutically acceptable salt of said compound or prodrug incombination with one or more anti-tumor agent.

The present application also provides pharmaceutical combinationcomposition comprising a therapeutically effective amount of acombination of a compound of formula (I) as described above, a prodrugthereof, or a pharmaceutically acceptable salt of said compound orprodrug; and one or more anti-tumor agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Identification of known compounds that block Ca2+ entry andproliferation and design of novel compounds with increased potency.Proliferation and Ca2+ entry were determined in Jurkat cancer cells asdescribed below in the Materials and Methods. Individualconcentration-response curves for each activity and compound wereconstructed and IC50 values were calculated. The calculated leastsquares regression in shown as a solid line and a line with a slope ofone in shown as a dashed line. Panel A: Using Ni2+ sensitivity as aguide, known compounds were identified that block Ca2+ entry andproliferation in Jurkat cells. Panel B: An SAR was developed from thedata depicted in panel A leading to the synthesis of novel compoundsthat block Ca2+ entry and proliferation Jurkat cells.

FIG. 2. The Ca2+ ionophore ionomycin overcomes the inhibition of Ca2+entry into and proliferation of Jurkat cells produced by TH-1177. PanelA. Ca2+ entry into Jurkat cells was determined as described in Materialsand Methods. Ionomycin at the indicated concentrations was added 30 sand the mitogenic monoclonal antibody OKT3 was added at a concentrationof 1 ug/ml at 60 s. Either EGTA at 2.5 mM or TH-1177 at the indicatedconcentrations was added at 150 s. The percentage inhibition wasdetermined as described in Material and Methods. Panel B. Jurkat cellswere grown for 48 hrs in the presence (open symbols) or absence (closedsymbols) of 30 nM ionomycin and the indicated concentrations of TH-1177.Percent control growth was determined as described in Materials andMethods.

FIG. 3. Amplicons of two sizes were identified in Jurkat and SK-N-SFcancer cell lines using α1H Ca2+ channel specific PCR primers. MessengerRNA was extracted and amplified as described in Materials and Methodsusing the primers described previously (Mariot, P., Vanoverberghe, K.,Lalevee, N., Rossier, M. F., and Prevarskaya, N. 2002. Overexpression ofan alpha 1H (Cav3.2) T-type calcium channel during neuroendocrinedifferentiation of human prostate cancer cells. J. Biol. Chem277:10824-10833). The resulting products were isolated by gelelectrophoresis and visualized by ethidium bromide staining andvisualized by UV illumination.

FIG. 4. The sequences of the amplicons shown in FIG. 4 (SEQ ID NOS: 1-4)are virtually identical to either α1H or its δ25 splice variant. Theamplicons shown in FIG. 4 were sequenced as described in Materials andMethods. The GenBank database was then queried and the alignments shownwere obtained.

FIG. 5. Blockade of the α1H Ca2+ current by the known T type Ca2+channel mibefradil or TH-1177 inhibits proliferation of stablytransfected HEK293 cells. HEK293 cells were stably transfected the α1Hgene as described in Materials and Methods. Proliferation and the Ca2+current were determined as described in Materials and Methods.

FIG. 6. TH-1177 and TH-1211 are stereoisomers about one of two chiralcenters and have different potencies at inhibiting the proliferation ofPC3 prostate cancer cells. Panel A: The structures of TH-1177 andTH-1211 were determined as described in Materials and Methods. Thediastereomers are racemic at the benzhydrol center (solid arrows) andenantiomeric at the proline center (open arrows) with TH-1177 having theS configuration and TH-1211 having the R. Panel B: The proliferation ofPC3 human prostate cancer cells was determined as described in Materialsand Methods. The IC50 for TH-1177 was 14 uM (open boxes) and for TH-1211was 42 uM (inverted triangles).

FIG. 7. TH-1177 and TH-1211 have different potencies at inhibiting theCa2+ current through transfected α1H channels. The current carried bytransfected α1H Ca2+ channels was determined as described in Materialsand Methods. The concentration response for TH-1177 and TH-1211 is shownin FIG. 6D. The IC50 for TH-1177 was 0.8 uM and for TH-1211 was 7 uM.

DEFINITIONS

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

As used herein, the term “purified” and like terms relate to anenrichment of a molecule or compound relative to other componentsnormally associated with the molecule or compound in a nativeenvironment. The term “purified” does not necessarily indicate thatcomplete purity of the particular molecule has been achieved during theprocess. A “highly purified” compound as used herein refers to acompound that is greater than 90% pure.

As used herein, the term “treating”, “treat” or “treatment” includesadministering therapy to prevent, cure, or alleviate/prevent thesymptoms associated with, a specific disorder, disease, injury orcondition. For example treating cancer includes inhibition or completegrowth arrest of a tumor, reduction in the number of tumor cells,reduction in tumor size, inhibition of tumor cell infiltration intoperipheral organs/tissues, inhibition of metastasis as well as relief,to some extent, of one or more symptoms associated with the disorder.The treatment of cancer also includes the administration of atherapeutic agent that directly decreases the pathology of tumor cells,or renders the tumor cells more susceptible to treatment by othertherapeutic agents, e.g., radiation and/or chemotherapy. As used herein,the term “treating” includes prophylaxis of the specific disorder orcondition, or alleviation of the symptoms associated with a specificdisorder or condition and/or preventing or eliminating said symptoms.

As used herein, the term “pharmaceutically acceptable carrier, vehicleor diluent” includes any of the standard pharmaceutical carriers, suchas a phosphate buffered saline solution, water, emulsions such as anoil/water or water/oil emulsion, and various types of wetting agents.The term also encompasses any of the agents approved by a regulatoryagency of the US Federal government or listed in the US Pharmacopeia foruse in animals, including humans.

The term “therapeutically effective amount” means an amount of acompound of the present invention that ameliorates, attenuates oreliminates a particular disease or condition or prevents or delays theonset of a particular disease or condition.

By “mammal” it is meant to refer to all mammals, including, for example,primates such as humans and monkeys. Examples of other mammals includedherein are rabbits, dogs, cats, cattle, goats, sheep and horses.Preferably, the mammal is a female or male human.

The phrase “compound(s) of the present invention” or “compound(s) ofFormula (I)” or the like, shall at all times be understood to includeall active forms of such compounds, including, for example, the freeform thereof, e.g., the free acid or base form, and also, all prodrugs,polymorphs, hydrates, solvates, tautomers, and the like, and allpharmaceutically acceptable salts, unless specifically stated otherwise.It will also be appreciated that suitable active metabolites of suchcompounds are within the scope of the present invention.

The expression “prodrug” refers to compounds that are drug precursorswhich following administration, release the drug in vivo via somechemical or physiological process (e.g., a prodrug on being brought tothe physiological pH or through enzyme action is converted to thedesired drug form).

The expression “pre-cancerous condition” refers to a growth that is notmalignant but is likely to become so if not treated. A “pre-cancerouscondition” is also known as “pre-malignant condition” by one of ordinaryskill in the art.

As used herein the term “anti-tumor agent” relates' to agents known inthe art that have been demonstrated to have utility for treatingneoplastic disease. For example, antitumor agents include, but are notlimited to, antibodies, toxins, chemotherapeutics, enzymes, cytokines,radionuclides, photodynamic agents, and angiogenesis inhibitors. Toxinsinclude ricin A chain, mutant Pseudomonas exotoxins, diphtheria toxoid,streptonigrin, boamycin, saporin, gelonin, and pokeweed antiviralprotein. Chemotherapeutics include 5-fluorouracil (5-FU), daunorubicin,cisplatinum, bleomycin, melphalan, taxol, tamoxifen, mitomycin-C, andmethotrexate as well as any of the compounds described in U.S. Pat. No.6,372,719 (the disclosure of which is incorporated herein by reference)as being chemotherapeutic agents. Radionuclides include radiometals.Photodynamic agents include porphyrins and their derivatives.Angiogenesis inhibitors are known in the art and include natural andsynthetic biomolecules such as paclitaxel, O-(chloroacetyl-carbamyl)fumagillol (“TNP-470” or “AGM 1470”), thrombospondin-1,thrombospondin-2, angiostatin, human chondrocyte-derived inhibitor ofangiogenesis (“hCHIAMP”), cartilage-derived angiogenic inhibitor,platelet factor-4, gro-beta, human interferon-inducible protein 10(“IP10”), interleukin 12, Ro 318220, tricyclodecane-9-yl xanthate(“D609”), irsogladine, 8,9-dihydroxy-7-methyl-benzo[b]quinoliziniumbromide (“GPA 1734”), medroxyprogesterone, a combination of heparin andcortisone, glucosidase inhibitors, genistein, thalidomide,diamino-anthraquinone, herbimycin, ursolic acid, and oleanolic acid.Anti-tumor therapy includes the administration of an anti-tumor agent orother therapy, such as radiation treatments, that has been reported asbeing useful for treating cancer.

As used herein, the term “halogen” or “halo” includes bromo, chloro,fluoro, and iodo.

The term “haloalkyl” as used herein refers to an alkyl radical bearingat least one halogen substituent, for example, chloromethyl, fluoroethylor trifluoromethyl and the like.

The term “C₁-C_(n) alkyl” wherein n is an integer, as used herein,represents a branched or linear alkyl group having from one to thespecified number of carbon atoms. Typically C₁-C₆ alkyl groups include,but are not limited to, methyl, ethyl, n-propyl, iso-propyl, butyl,iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.

The term “C₂-C_(n) alkenyl” wherein n is an integer, as used herein,represents an olefinically unsaturated branched or linear group havingfrom 2 to the specified number of carbon atoms and at least one doublebond. Examples of such groups include, but are not limited to,1-propenyl, 2-propenyl, 1,3-butadienyl, 1-butenyl, hexenyl, pentenyl,and the like.

The term “C₂-C_(n) alkynyl” wherein n is an integer refers to anunsaturated branched or linear group having from 2 to the specifiednumber of carbon atoms and at least one triple bond. Examples of suchgroups include, but are not limited to, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 1-pentynyl, and the like.

The term “C₃-C_(n) cycloalkyl” wherein n=4-8, represents cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

As used herein, the term “optionally substituted” refers to from zero tofour substituents, wherein the substituents are each independentlyselected. Each of the independently selected substituents may be thesame or different than other substituents.

As used herein the term “aryl” refers to a mono- or bicyclic carbocyclicring system having one or two aromatic rings including, but not limitedto, phenyl, benzyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, andthe like. Substituted aryl includes aryl compounds having one or twoC₁-C₆ alkyl, halo or amino substituents. The term (C₅-C₈ alkyl)arylrefers to any aryl group which is attached to the parent moiety via thealkyl group. The term “heterocyclic group” refers to a C₃-C₈ cycloalkylgroup containing from one to three heteroatoms wherein the heteroatomsare selected from the group consisting of oxygen, suilfur, and nitrogen.

The term “bicyclic” represents either an unsaturated or saturated stable7- to 12-membered bridged or fused bicyclic carbon ring. The bicyclicring may be attached at any carbon atom which affords a stablestructure. The term includes, but is not limited to, naphthyl,dicyclohexyl, dicyclohexenyl, and the like. Any ring structure drawnwith one or more bonds extending from the center of the ring is intendedto designate a series of compounds that have a bond(s) extending fromone of the carbon atoms of the ring to another atom. For example, thestructure:

designates a series of compounds including 2-thienyl or 3-thienyl groupsthat contain an R₇ substituent at one of the remaining ring carbonatoms.

The compounds of the present invention contain one or more asymmetriccenters in the molecule. In accordance with the present invention astructure that does not designate the stereochemistry is to beunderstood as embracing all the various optical isomers, as well asracemic mixtures thereof.

The term “pharmaceutically acceptable salt” refers to salts which retainthe biological effectiveness and properties of the compounds of thepresent invention and which are not biologically or otherwiseundesirable. In many cases, the compounds of the present invention arecapable of forming acid and/or base salts by virtue of the presence ofamino and/or carboxyl groups or groups similar thereto.

It is understood to one skilled in the art that “T type calcium channelinhibitors” are also known as “T type calcium channel inhibitors”.

DETAILED DESCRIPTION OF THE INVENTION

Changes in the cytosolic concentration of calcium are a criticalcomponent of numerous cellular processes. The calcium necessary forthese changes comes from the extracellular milieu via influx throughcalcium channels. Calcium channels are grouped into several familiesbased upon sequence analysis, biophysical characteristics andpharmacological sensitivity. Among these is the T type calcium channelfamily. These calcium channels have been implicated in regulation ofblood pressure, cardiac rhythm, and cellular proliferation. Howeverthere has been only one pharmacological agent, mibefradil, that has beenproven to be clinically effective because of inhibition of T channelfunction. Unfortunately, mibefradil was withdrawn from market due toadverse interactions with other drugs leaving this therapeutic approachunavailable.

One embodiment of the present invention is directed to a series of novelsynthetic compounds that inhibit calcium entry via T type calciumchannels while other members of this series activate calcium entry.Inhibitors of calcium entry will be useful for treating hypertension,cardiac arrhythmia and clinically deleterious cellular proliferationwhile activators of calcium entry will be useful as cardiac stimulantsand inducers of programmed cell death with the intent of reducing theburden of clinically unwanted cells such as cancer cells. Examples ofthese compounds are as follows:

Exhibit A Presence of Oxygen Atom

TABLE 5.1 Jurkat PC3 LNCaP MDA-435 MDA-231 MDA-361 prolif Ca2+ prolifCa2+ prolif Ca2+ prolif Ca2+ prolif Ca2+ prolif Ca2+ Series 1 3.1 4 1011 NE 3.5 10 12 25 8 10 8 10 3.2 3 3  9  10 2  5 10.5 10 10 8 4 6 Series2 3.3 70 60 >100 NE 42 NE >100 >100 100 3.4 3.7 3 10 ~15 5.5 15 10 15 4~20  4 10 Series 1

Series 2

NE = no effect at highest concentration of drug tested, all values inmicromolar In all Figures, “prolif” indicates the IC₅₀ for inhibition ofproliferation of the indicated cell lines while “Ca2+” is the IC₅₀ forinhibition of calcium entry. As can be seen in this Figure, the presenceof an ether linkage has a profound effect of biological activity butonly when the amine is proximal to it.

Presence of Nitrogen

TABLE 5.2 Jurkat PC3 LNCaP MDA-435 MDA-231 MDA-361 prolif Ca2+ prolifCa2+ prolif Ca2+ prolif Ca2+ prolif Ca2+ prolif Ca2+ Series 1 3.5 50 733 30 25 10 65 10 50 10 45 25 3.6 >100 >100 >100 NE ~100 NE >100 NE >100NE >100 NE 3.8 7.5 10 11.5 10 3.5 10 15.7 10 10.6 10 7.6 20 Series 2 3.7~100 10 NE NE ~100 NE NE NE >100 60 >100 NE 3.9 3.8 3 4 NE 1.6  3 12 1011.7 10 4  5 Series 1

Series 2

NE = no effect at the highest concentration of drug tested, all valuesin micromolar This Figure demonstrates that nitrogen in the hydrocarbon“tether” is required for biological activity.

Basicity of the Nitrogen

TABLE 5.3 Jurkat PC3 LNCaP MDA-435 MDA-231 MDA-361 prolif Ca2+ prolifCa2+ prolif Ca2+ prolif Ca2+ prolif Ca2+ prolif Ca2+ Series 1 3.10 >300NE >1 mM NE >1 mM NE >1 mM NE >1 mM NE ~70 NE 3.11 5.4 3.3 14 NE 3.3 NE22 NE 11.6 20 8.5 10 Series 2 3.14 >100 30 >100 NE >100 30 NE 30 NE 30NE 10 3.15 4 10 11 NE 3.5 10 12 25 8 10 8 10 Series 1

Series 2

NE = no effect at the highest concentration of drug tested, all valuesin micromolar This Figure demonstrates that the basicity of the nitrogencontributes to the biological activity.

Steric Bulk Around the Nitrogen

TABLE 5.4

Jurkat PC3 LNCaP MDA-435 MDA-231 MDA-361 Compound prolif Ca2+ prolifCa2+ prolif Ca2+ prolif Ca2+ prolif Ca2+ prolif Ca2+ 3.14 7.6 10 11.5 103.5 10 15.7 10 10.6 10 7.6 20 3.15 12 10 32 20 4 7 27 20 24 10 16 203.16 23 30 55 NE 33 10 50 NE 21 NE 8 NE

NE = no effect at the highest concentration of drug tested, all valuesin micromolar From this Figure, it can be seen that steric bulk aroundthe nitrogen decreases biological activity.

Location of the Nitrogen

TABLE 5.5 Nitrogen Oxygen Jurkat PC3 LNCaP MDA-435 MDA-231 CompoundPosition Present prolif Ca2+ prolif Ca2+ prolif Ca2+ prolif Ca2+ prolifCa2+ 3.17 1 no >100 30 >100 NE >100 30 NE 30 NE 30 3.1 2 no 4 10 11 NE3.5 10 12 25 8 10 3.19 3 no 4 10 10 NE 3 10 11 10 6 10 3.3 4 no 7060 >100 NE 42 NE >100 >100 100 3.21 5 no 30 30 11 30 7 NE 9 15 30 3.22 6no 13 3 9 NE 9 NE 40 NE 25 10 3.23 7 no ~30 NE >100 NE ~100 NE NE NE NENE

NE = no effet at the highest concentration of drug tested, all values inmicromolar This Figure shows that biological activity is affected by theposition of the nitrogen along the hydrocarbon tether.

In accordance with one embodiment a novel compound that inhibit Ca2+entry, and thereby proliferation of cancer cells is provided. Thecompounds have the general structure of Formula (I):

wherein

-   -   R₁ is C₁-C₄ alkyl, hydroxy, or C₁-C₄ alkoxy;    -   Z is NH, NCH₃, O, S, or CH₂;    -   Y is NH, O, or CH₂ with the proviso that Y and Z are not the        same;    -   R₂ is H, halo, NH₂, C₁-C₄ alkyl, hydroxy, or C₁-C₄ alkoxy;    -   m and n are independently selected from integers ranging from        1-5 with the proviso that m+n=an integer ranging from 2-9; and    -   R₃ is H, halo, NH₂, C₁-C₄ alkyl, hydroxy, or C₁-C₄ alkoxy;    -   or a pharmaceutically acceptable salt thereof.

Preferably, R₁ is hydroxy or C₁-C₄ alkoxy; Z is N or O; R₂ is H, halo,NH₂ or hydroxy; and R₃ is H.

The novel compounds of the present invention can be combined withstandard pharmaceutically acceptable carriers or other known anti-tumorand chemotherapeutic agents.

Materials and Methods

Synthesis of TH-1177:

TH-1177 was synthesized in three simple steps as described (Haverstick,D. M., Heady, T. N., Macdonald, T. L., and Gray, L. S. 2000. Inhibitionof human prostate cancer proliferation in vitro and in a mouse model bya compound synthesized to block Ca²⁺ entry. Cancer Res 60:1002-1008).L-Proline methyl ester was coupled with 4-methoxyphenylacetic acid usingbenzotriazol-1-yl-oxytripyrrolidinophosphonium to generate methyl1-[2-(4 methoxyphenyl)acetyl]pyrrolidine-2-carboxylate, a yellowish oil.The resulting amide was subsequently reduced to the amino alcohol withLiAIH4 and AICI3 in tetrahydrofuran. The resulting colorless oil wascoupled with 4-chlorobenzhydrol under Williamson conditions withcatalytic p-toluenesulfonic acid in refluxing toluene. The finalbrownish oil was isolated by column chromatography, and its structurewas confirmed by nuclear magnetic resonance and mass spectrometry.TH-1177 was dissolved in DMSO for use.

Cell Lines and Maintenance:

Cancer cell lines were obtained from the American Type CultureCollection (Manassas, Va.). Cell lines were maintained in RPMI1640supplemented with glutamine and 5% fetal bovine serum containingSerXtend (Irvine Scientific) when appropriate insulin, as directed bythe ATCC. The fetal bovine serum used for culture was heat-inactivatedby maintaining the serum at 56° C. for 1 h. Cell lines used were a Tcelleukemia (Jurkat), prostate cancer (PC3, DU145, LNCAP), breast cancer(MDA468, MDA361, MCF7), pancreatic cancer (MIA PaCa2), liver cancer (HepG2), lung cancer (A549, NCI H460), colon cancer (HT29, HCT 116), andovarian cancer (SK-OV-3).

Measurement of the [Ca2+]i Concentration:

Cells were incubated in growth media containing 1 uM of theacetoxy-methyl ester of the Ca2+-sensitive fluorescent dye indo-1(indo-1/AM; Molecular Probes, Eugene, Oreg.) for 1 h at 37° C. Cellswere washed three times in buffer A [10 mM HEPES (pH 7.4), 1 mM MgCl₂, 3mM KCl, 1 mM CaCl₂, 140 mM NaCl, 0.1% glucose, and 1% fetal bovineserum] and suspended to a final concentration of 10⁶ cells/ml. Beforestimulation, cells were warmed to 37° C. Changes in [Ca2+]i weremonitored in an SLM 8100C spectrofluorimeter (SLM/Aminco; Urbana, Ill.)using previously published methods (Densmore, J. J., Haverstick, D. M.,Szabo, G., and Gray, L. S. 1996. A voltage operable current is involvedin activation-induced Ca²⁺ entry in human lymphocytes whereas I_(CRAC)has no apparent role. Am. J. Physiol. 271:C1494-C1503; Haverstick, D.M., Densmore, J. J., and Gray, L. S. 1998. Calmodulin regulation of Ca²⁺entry in Jurkat T cells. Cell Calcium 23:361-368).

Measurement of Cellular Proliferation:

LNCaP cells at 2.5×10⁴ cells/well or PC-3 cells at 5×10⁴ cells/well,both in a final volume of 100 I.JI, were plated in triplicate instandard flat-bottomed 96-well tissue culture plates in the presence ofdrug or vehicle (DMSO). Unless otherwise indicated, cells were grown for48 h at 37° C. in a CO₂ incubator. Relative cell growth was determinedwith the CellTiter 96 aqueous cell proliferation assay (Promega,Madison, Wis.) as described by the manufacturer using an automated platereader. Results were calculated in a blinded fashion and are the meansof triplicate determinations.

Results

Construction of a Novel Chemical Library:

Extracellular Ni2+ blocks the Ca2+ entry pathway in electricallynon-excitable cells (Merritt, J. E., Jacob, R., and Hallam, T. J. 1989.Use of manganese to discriminate between calcium influx and mobilizationfrom internal stores in stimulated human neutrophils. J. Biol. Chem.264:1522-1527; Jones, G. R. N. 1985. Cancer therapy: Phenothiazines inan unexpected role. Tumori 71:563-569) as well as the current through Ttype Ca2+ channels (Lee, J.-H., Gomora, J. C., Cribbs, L. L., andPerez-Reyes, E. 2000. Nickel block of three cloned T-type Ca channels:low concentrations selectively block α1H. Biophys. J. 77:3042). We madeuse of these facts and conducted a search of the Medline database forcompounds that block Ca2+ entry in any system that was also sensitive toinhibition of Ca2+ entry by Ni2+. The identified compounds were thenused as the basis for a reiterated search. This strategy was continueduntil the only citations returned were those that had been retrievedalready indicating that the database had been saturated. These agents,some of which are listed in Table 1, were tested for the ability toblock proliferation of and Ca2+ entry into the Jurkat human cancer cellline. These compounds were tested in various cancer cell lines(Materials and Methods) with results similar to those obtained with theJurkat cell line (data not shown). The correlation between these twoinhibitory activities in the Jurkat cell line, expressed as ICso's, isshown in FIG. 1, panel A. The resulting structure-activity relationship(SAR) was used as a guide to synthesize novel chemical agents. Thesenovel compounds exhibited enhanced inhibition of Ca2+ into andproliferation (Table 2). The slope of the regression line between theability of the novel compounds to inhibit proliferation and block Ca2+entry was 0.97 or very close to unity with an r² value of 0.93 (FIG. 1,panel B) compared to a slope of 0.73 (r²=0.79) for the known agents(FIG. 1, panel A). Because Ca2+ entry is required for proliferation(Berridge, M. J., Lipp, P., and Bootman, M. D. 2000. The versatility anduniversality of calcium signalling. Nat. Rev. Mol Cell Biol. 1:11-21),the slope of 0.97 should most appropriately be interpreted in a Bayesianfashion. This Bayesian analysis suggests that all of the effect of thesecompounds on proliferation is mediated through inhibition of Ca2+ entry.

The Ca2+ ionophore ionomycin partially overcomes the effects of TH-1177.We have used one of our compounds, TH-1177, as the prototype for theothers (Haverstick, D. M., Heady, T. N., Macdonald, T. L., and Gray, L.S. 2000. Inhibition of human prostate cancer proliferation in vitro andin a mouse model by a compound synthesized to block Ca²⁺ entry. CancerRes 60:1002-1008). If TH-1177 is acting via inhibition of Ca2+ entry,its effects should be at least partially reversed by direct elevation of[Ca2+]i using a Ca2+ ionophore. As shown in FIG. 2, panel A, ionomycinovercame inhibition of Ca2+ entry by TH-1177 in a concentrationdependent manner although there was no effect on proliferation of 30 nMionomycin alone. Ionomycin also reduced the ability of TH-1177 toinhibit proliferation (FIG. 2, panel B) increasing the IC50 of TH-1177from 4.6 uM in the presence of 30 uM ionomycin to 17.8 uM in itsabsence. This suggests that TH-1177 is acting to inhibit proliferationby inhibition of Ca2+ entry and is in accord with the relationshipbetween Ca2+ entry and proliferation shown in FIG. 1.

Cancer cell lines sensitive to our agents express message for the α1HCa2+ channel or its δ25 splice variant.

We have presented data previously suggesting that a member or members ofthe T type Ca2+ channel family have a role in mediating Ca2+ entry inelectrically excitable cells (Densmore, J. J., Haverstick, D. M., Szabo,G., and Gray, L. S. 1996. A voltage operable current is involved inactivation-induced Ca²⁺ entry in human lymphocytes whereas I_(CRAC) hasno apparent role. Am. J. Physiol. 271:C1494-C1503; Densmore, J. J.,Szabo, G., and Gray, L. S. 1992. A voltage-gated calcium channel islinked to the antigen receptor in Jurkat T lymphocytes. FEBS Lett.312:161-164; Haverstick, D. M. and Gray, L. S. Increased intracellularCa²⁺ induces Ca²⁺ influx in human T lymphocytes. Molecular Biology ofthe Cell 4, 173-184. 1993; Haverstick, D. M., Densmore, J. J., and Gray,L. S. 1998. Calmodulin regulation of Ca²⁺ entry in Jurkat T cells. CellCalcium 23:361-368). It has been shown recently that a prostate cancerline expresses the α1H isoform of T type Ca2+ channels at levels thatvary with differentiation status (Mariot, P., Vanoverberghe, K.,Lalevee, N., Rossier, M. F., and Prevarskaya, N. 2002. Overexpression ofan alpha 1H (Cav3.2) T-type calcium channel during neuroendocrinedifferentiation of human prostate cancer cells. J. Biol. Chem277:10824-10833). Using the same primers, reported in that study, thevalue for TH-1211 is 24 uM. Thus, when measured by Ca2+ selectivefluorescent dyes, Ca2+ entry was similarly sensitive to TH-1177 andTH-1211 as was the Ca2+ current mediated by α1H. As importantly, each ofthese measures of Ca2+ influx showed the same relative difference insensitivity to the stereoisomers. This shows the pharmacologicalcorrespondence between capacitative Ca2+ entry when measured byfluorescence and Ca-2+ entry mediated by α1H when measured byelectrophysiological methods.

Discussion

Recently, several channels have been suggested as candidates formediating Ca2+ entry in electrically non-excitable cells. A commonfeature of these proposed Ca2+ entry channels is that they are notvoltage gated. This reflects the prevalent assumption that the Ca2+entry pathways in electrically excitable and non-excitable cells arecategorically distinct. It is extremely difficult, however, tounderstand why such an assumption is necessary particularly in view ofthe inherently non-categorical nature of biological systems. Inaddition, these voltage insensitive Ca2+ channels do not fulfillcompletely the biophysical criteria for the capacitative Ca2+ entrypathway in electrically non-excitable cells (Haverstick, D. M. and Gray,L. S. Increased intracellular Ca²⁺ induces Ca²⁺ influx in human Tlymphocytes. Molecular Biology of the Cell 4, 173-184.1993).

This uncertainty about the nature of Ca2+ entry in electricallynon-excitable cells may be the result of a number of technical factorsin addition to the constraints imposed by unnecessary assumption. Whileseveral of the proposed candidates for mediation of Ca2+ entry inelectrically non-excitable cells have been characterized at themolecular level (Putney, J. W., Jr. and McKay, R. R. 1999. Capacitativecalcium entry channels. Bioessays 21:38-46), a commonly acceptedCandidate, I_(CRAC) (Clapham, D. E. 2002. Sorting out MIC, TRP, and CRACIon Channels. J. Gen. Physiol 120:217-220; Cahalan, M. D., Wulff, H.,and Chandy, K. G. 2001. Molecular properties and physiological roles ofion channels in the immune system. J Clin Immunol 21:235-252), has not.It is also difficult to tie the function of these channels to inhibitionof proliferation of cancer cell lines because of the paucity of specificCa2+ entry inhibitors for electrically non-excitable cells. There is aswell the unavoidable disjunction between Ca2+ entry as measured by Ca2+selective fluorescent dyes and electrophysiological methods: Thepatch-ctamp technique is extraordinarily powerful for examining thebiophysical details of the function of an ion channel (Neher, E. andSakmann, B. 1992. The patch clamp technique. Scientific AmericanMarch:44-51), however the level of membrane control it both achieves andrequires makes it less suited to identifying a channel's physiologicalrole. Fluorescence techniques are very limited in obtaining biophysicaldetail but better able to study physiological roles. This disconnectionmakes it difficult to determine if the effects of physiologicallyrelevant stimuli, as determined by fluorescence measurements, arereproduced at the electrophysiological level.

We have suggested that the mechanism of Ca2+ entry in electricallynon-excitable cells involves a Ca2+ channel sharing characteristics withthe T type family of voltage gated Ca2+ channels (Densmore, J. J.,Haverstick, D. M., Szabo, G., and Gray, L. S. 1996. A voltage operablecurrent is involved in activation-induced Ca²⁺ entry in humanlymphocytes whereas I_(CRAC) has no apparent role. Am. J Physiol.271:C1494-C1503; Densmore, J. J., Szabo, G., and Gray, L. S. 1992. Avoltage-gated calcium channel is linked to the antigen receptor inJurkat T lymphocytes. FEBS Lett. 312:161-164; Haverstick, D. M. andGray, L. S. Increased intracellular Ca²⁺ induces Ca²⁺ influx in human Tlymphocytes. Molecular Biology of the Cell 4, 173-184. 1993; Haverstick,D. M., Densmore, J. J., and Gray, L. S. 1998. Calmodulin regulation ofCa²⁺ entry in Jurkat T cells. Cell Calcium 23:361-368). It could beargued that it is difficult to envision a physiologic role for voltagegated Ca2+ channels in cells that do not have action potentials. Thisargument is, however, based upon the assumption that a voltage gatedCa2+ channel can be only be activated by an action potential. Such anassumption is false a priori because the means by which a protein can beregulated by imposed experimental conditions is not necessarilyidentical with, or even similar to, the mechanism by which it iscontrolled physiologically. Although secondary to regulation by membranepotential, the known biochemical regulation of voltage gated Ca2+channels in a variety of systems also suggests that the categoricaldistinction between electrical and biochemical regulation of Ca2+channels may be simplistic.

The data presented here strongly suggest the possibility that the α1Hisoform of nominally voltage gated T type Ca2+ channels or its 825splice variant has a role in Ca2+ entry into and proliferation ofelectrically non-excitable cancer cells. Our data show that novelcompounds can be created based upon an structure-activity relationshipgenerated from compounds that are known to inhibit Ca2+ entry in systemsthat are also sensitive to inhibition by Ni2+. These novel compoundsalso inhibit proliferation of several cancer cell lines via blockade ofCa2+ entry. The cell lines that are sensitive to our agents expressmessage for α1H Ca2+ channels, its δ25 splice variant, or both. Thesecompounds also inhibit the Ca2+ current mediated by α1H Ca2+ channelsand slow the proliferation of HBEK293 cells stably transfected with theα1H Ca2+ channel isoform. TH-1177 and TH-1211 stereoselectively inhibitCa2+ entry into and proliferation of cancer cell lines and show thesame-stereosetectivity and potency in blocking canonical α1H. These datastrongly suggest that the α1H Ca2+ channel and its δ25 splice variantparticipate in Ca2+ entry in the cancer cell lines tested in thesestudies.

Linking biophysical analysis of Ca2+ channel function to a physiologicalfunction such as proliferation can pose challenges. We have demonstratedthat our compounds block a heterologously expressed Ca2+ channel andthat only those cancer cell lines with message for that channel, or itssplice variant, are sensitive to inhibition by the same agents.Furthermore, TH-1177 is more potent at inhibiting Ca2+ entry viaexpressed α1H as measured by biophysical techniques than thestereoisomer of it, TH-1211. TH-1177 and TH-1211 also show the same rankorder of potency at inhibiting proliferation and Ca2+ entry in cancercell lines when these are assayed by more commonly used biochemicalmethods. The absolute potencies of the agents as measured by IC50 valuesare strikingly similar whether measured by biophysical or biochemicalmethods. Thus, the results from a combination of experimental approacheswere synthesized into a picture of the likely mechanism of Ca2+ entry insome cancer cells.

Expression of the α1H Ca2+ channel has been demonstrated in LNCaP cellsand the expression level correlates with differentiation state (Mariot,P., Vanoverberghe, K., Lalevee, N., Rossier, M. F., and Prevarskaya, N.2002. Overexpression of an alpha 1H (Cav3.2) T-type calcium channelduring neuroendocrine differentiation of human prostate cancer cells. JBiol. Chem 277:10824-10833). Although the sequence of the δ25 splicevariant has been deposited in GenBank (accession number AF223563), itsfunction has not been described to our knowledge. However, both aremembers of the T type Ca2+ channel family by sequence homology and havebeen assigned to the Hs.122359 UniGene cluster within the NCBI database.The physiological roles of T type Ca2+ channels are not wholly clear atpresent although they may playa role as pacemakers in the heart andcentral nervous system (Chemin, J., Monteil, A., Perez-Reyes, E.,Bourinet, E., Nargeot, J., and Lory, P. 2002. Specific contribution ofhuman T-type calcium channel isotypes (α_(1G), α_(1H) and α_(1I)) toneuronal excitability. J. Physiol. (Lond.) 540:3-14; McDonald, T. F.,Pelzer, S., Trautwein, W., and Pelzer, D. J. 1994. Regulation andmodulation of calcium channels in cardiac, skeletal, and smooth musclecells. Physiol Rev 74:365-507). The expression of these Ca2+ channelsalso appears to be developmentally regulated (Brooks, G., Harper, J. V.,Bates, S. E., Haworth, R. S., Cribbs, L. L., Perez-Reyes, E., andShattock, M. J. 1999. Over expression of the voltage-gated T-typecalcium channel induces vascular smooth muscle cell proliferation.Circulation 100:1-209(Abstr.); Clozel, J. P., Ertel, E. A., and Ertel,S. I. 1999. Voltage-gated T-type Ca²⁺ channels and heart failure. Proc.Assoc. Am Physicians 111:429-437; Harper, J. V., McLatchie, L.,Perez-Reyes, E., Cribbs, L. L., Shattock, M. J., and Brooks, G. 2000.T-type calcium channel expression is necessary for G1-S progression invascular smooth muscle. Circulation 102:II-48 (Abstr.); Monteil, A.,Chemin, J., Bourinet, E., Mennessier, G., Lory, P., and Nargeot, J.2000. Molecular and functional properties of the human α_(1G) subunitthat forms T-type calcium channels. J. Biol. Chem. 275:6090-6100) andthe data reported here suggest that both canonical α1H and its δ25splice variant are responsible for the Ca2+ entry required forproliferation of some cancer cell lines. These data are also consistentwith the idea that the so-called voltage gated C12+ channels are likelyto have a physiologically role in cell types conventionally categorizedas electrically non-excitable.

Our novel synthetic compounds may have clinical utility becausetreatment of mice bearing xenografted human PC3 prostate cancer cellswith TH-1177 significantly extended the lifespan of them (Haverstick, D.M., Heady, T. N., Macdonald, T. L., and Gray, L. S. 2000. Inhibition ofhuman prostate cancer proliferation in vitro and in a mouse model by acompound synthesized to block Ca²⁺ entry. Cancer Res 60:1002-1008).Thus, it is possible that Ca2+ channel inhibitors will ultimatelyprovide clinicians with an addition to their armamentarium for thetreatment of cancer. The observations presented here, as well as thosepresented previously (Haverstick, D. M., Heady, T. N., Macdonald, T. L.,and Gray, L. S. 2000. Inhibition of human prostate cancer proliferationin vitro and in a mouse model by a compound synthesized to block Ca²⁺entry. Cancer Res 60:1002-1008), lay the groundwork for furtherdevelopments in this area.

What is claimed is:
 1. A compound of structural formula

wherein R₁ is hydroxy, or C₁-C₄ alkoxy; Z is NH, NCH₃, O, or S; Y isCH₂; R₂ is H, halo, NH₂, C₁-C₄ alkyl, hydroxy, or C₁-C₄ alkoxy; m and nare independently selected from integers ranging from 1-5 with theproviso that m+ n=an integer ranging from 2-9; and R₃ is chloro; or apharmaceutically acceptable salt thereof.
 2. The compound according toclaim 1, wherein R₁ is hydroxy or C₁-C₄ alkoxy; Z is NH, NCH₃ or O; andR₂ is H, halo, NH₂ or hydroxy.
 3. A compound selected from the groupconsisting of the following compounds:


4. A pharmaceutical composition comprising a therapeutically effectiveamount of a compound of claim 1 or a compound of claim 3, or apharmaceutically acceptable salt of said compound; and apharmaceutically acceptable carrier, vehicle or diluent.
 5. Apharmaceutical combine a compound of claim 1 or a compound of claim 3,or a pharmaceutically acceptable salt of said compound therapeuticallyeffective amount of a combination of a compound of formula (I) asdescribed in; and one or more anti-tumor agents.
 6. The compound ofclaim 1 or 2 wherein R₁ is (C₁-C₄)alkoxy.